Viable deletion mutant in the medium and large T-antigen-coding sequences of the polyoma virus genome.

0022-538X/80/03-1215/06$02.00/0

Viable Deletion

Mutant in the Medium and

Large

T-Antigen-Coding

Sequences

of the

Polyoma

Virus Genome

MARY M.BENDIG,tTHRESIATHOMAS,ANDWILLIAMR. FOLK*

Departmentof Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48109

Apolyomavirusmutantthat maps intheearly regionbetweenthe known

hr-tandts-amutantshasbeen isolated. Its66-base-pair deletion resultsinstructural

changes in both medium andlarge T-antigensbutcauses nosubstantial alterations

inviralreplicationorcelltransformation.

Theearly region of thepolyomavirusgenome

contains two known complementation groups

defined by mutants, the

host-range-nontrans-forming (hr-t) mutants and the

temperature-sensitivets-a mutants.Thehr-tmutantsmapin

the proximal portion of the early region. They

induce analteredhost rangeforlyticgrowthand

are defective in virus-induced cell transforma-tion andtumorigenesis(3, 8). Thets-amutants

mapin thedistalportionof theearly regionand

aredefective,atthenonpermissivetemperature,

inDNAreplication, control of viral RNA

tran-scription, and virus-inducedcelltransformation

(6,7, 9, 10,25,26).Inthisreportwedescribe the

isolation andpartial characterization ofa

dele-tionmutant, mutant45,thatmaps intheearly

region between the hr-t andts-amutants.

Mutant45 was detected while screening for

mutants with deletions at the endo BglI site

neartheorigin ofreplication.Theisolation

pro-cedure has beendescribed elsewhere(1).Inbrief,

form I DNA from unmutagenized Pasadena large-plaque polyoma virus was digested with

endo BglI (which cuts polyoma DNA at one

site) and then electrophoresed through a 1%

agarose gel.Theresultingform IIIlinearDNA

was extracted from the gel and digested first withSi nuclease (14 U ofenzyme per 2.5

,jg

of

DNAfor30minat

25°0)

and then with

exonu-clease m (0.4 U ofenzyme per 1.2 ,ugof DNA

for 60 min at 3700). Secondary whole mouse

embryo cells were infected with the digested DNA, and fromtheresulting virus stock

individ-ual plaques werepicked and tested for

altera-tions in their endo HpaH digestion patterns.

Mutant45gave awild-typesizeHpaII fragment

5 (HpaII-5contains the endoBglIsite, Fig. 1)

but wasmissingwild-typesizeHpaII fragments

4and 8. Anewfragment slightlysmallerin size

than that expected of a composite HpaII-4/8

fragmentwasobserved (Fig. 2), thussuggesting

that mutant 45 has adeletionremoving the endo

HpaII-4/8 site. The altered virus was plaque

t Present address: Institut fir Molekularbiologie H der Universitit Ziirich, 8093 Ziirich, Switzerland.

purifiedonce morebeforebeing further

charac-terized.

Digestion ofmutant45DNAwith

endonucle-asesAluI (4) and HaellI (12) indicated that a

60- to 100-base-pair deletion had occurred

be-tween(butnotincluding)theAvaI site and the

PvuII site locatedoneitherside of the

Hpall-4/

8 junction. Accordingly, mutant 45 DNA was

cleaved with endonucleaseAvaIand incubated

FIG. 1. Map of the polyoma virus genome. The circular map is divided into the the eight HpaII

fragments (12). The single endo BglIsite and the

endoAvaI and endo PvuIIsites mapping near the

endoHpaII-4/8siteareindicated by arrows (11). On

the outeredge of the map, dotted lines define the locationof the hr-tmutants(14), the ts-a mutants (25), andmutant45;solidlinesdefine thepredicted coding regionsfor the small, medium, and large T-antigens (11, 30; R. Kamen, personal communication). On the inside ofthe map, theorigin of replication and the earlyandlate regions of the genome are indicated

(19, 21).

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A B MUTANT 45

G A+G C+T C A+C

Hpal1- I-- ow

4

2 * _

3-->-M.w

- -HpolI-4/8

4

6---6.... .. i..0

C

Ac

A G

8-*

FIG. 2. Autoradiogramofpolyoma DNA digested

with endoHpaII. EndoHpaIIdigests of[32P/DNA from the wild type(slotA) andmutant 45 (slotB)

wereanalyzed byelectrophoresisina6%

polyacryl-amidegel(2).Arrowsontheleftpointtothewild-type

HpaIIfragments. Thearrow onthe rightpoints to

thecomposite HpaII-4/8fragment foundinmutant

45.

with[y-32P]ATPandpolynucleotide kinase and

sequenced by the chemical degradation

proce-dure ofMaxamand Gilbert(24).Comparisonof

its sequence with that of the wild-type parent

revealed that a 66-nucleotide deletion had

oc-curred between nucleotides 1090 and 1157

(Friedmannnumberingsystem[11]; Fig. 3).

Be-cause the basesdeletedare amultipleofthree,

thereadingframe isnotaltered.

Inspectionof the sequence in thisregion

sug-gests apossiblereasonfor the susceptibilityof

polyomaDNAtoS1nuclease. Between

nucleo-tides 1132and1183 occursapartialpalindrome

which could formahairpin (Fig. 4).Thismight

providearegionofsingle-strandedDNA

suscep-tibletoS1nuclease.

Theearly regionof thepolyomavirus genome

GG

TC AA

GG G

Tc

TA AA

Gc

FIG. 3. AutoradiogramofpolyomaDNAsubjected

tochemicaldegradationandgelelectrophoresis. Pol-yomaDNAs(mutant45anditswild-type parent)were

cloned in Escherichia coliwithpBR322(Bendiget

al.,manuscriptinpreparation).EndonucleaseAvaI

digestionofthepurifiedchimericDNAgenerateda

fragment containing the majority of the polyoma

earlyregion. Thiswaslabeled with[y-32P]ATPand

polynucleotidekinase andthendigestedwith

endo-nucleaseEcoRI. Theasymmetrically labeled DNAs

wereseparatedandsubjectedtochemical

degrada-tion (10min). Theproducts wereseparatedon 15% polyacrylamidegels.

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I06 SMutant 45

CCT TGT GTT GCT GAG CCC GAT GACAGCATATCCI CCCC CCC AGA ACT CCT GTA TCCAGA

LargeT-antigen PRO CYS-VAL ALA-GLU PRO ASP ASP-SER*ILE-SER-PRO-ASP-PROPRO-ARG-THR-PRO-VAL-SER-ARG MiddleT-antigen ILE-LEU VAL LEU LEU SER-PRO MET-THR-ALA-TYR-PRO ARGTHR-PRO-PRO-GLU-LEU-LEU-TYR-PRO GLU

1194

AAS CGA CCA AGACC8ICGGA GCCACTGOA GGA GGA GGAGGA GGA GTA

LYS ARG-PRO-ARG-PRO ALAGLY*ALA-THR-GLY GLY GLY*GLYGLY*GLY VAL

[image:3.504.45.442.61.162.2]

SER-ASP-GLN-ASP-GLNMLEU-GLU-PRO-LEU*GLU-GLU*GLU-GLU-GLU-GLUTYR

FIG. 4. Sequence of the region of wild-type polyoma DNA including themutant45deletion. The nucleotide sequenceofthe parentofmutant45wasincompleteagreement with that determinedbyFriedmannetal.(11)

andSoeda etal. (30).Arrowspointto theregion deleted in mutant45. Underlined sequences indicatea

partial palindrome which couldformanintrastrandloop. The HpaII-4/8 and PvuII sites indicated inFig.1 areenclosed in boxes. Amino acid sequencesformiddle andlargeT-antigensareindicated(11, 30).

codes for at least three tumor antigens of

ap-proximately95,000(95K), 60K, and22K molec-ularweight (15,16,18,29).In ts-amutants,large

T-antigen (95K) isthermolabile, whereas the60

and 22KT-antigens remain unaffected (16, 18,

26,29).Incontrast,the hr-t mutationsaffect the

60and22Kantigens butnotthe 95KT-antigen

(16, 17, 27, 29). To determine whether mutant

45synthesizedearly viral proteins of normal size

andamounts, 3T6 cellswere infected, the

pro-teinswerelabeled with

['S]methionine,

and the T-antigens were immunoprecipitated and

ana-lyzed by sodiumdodecylsulfate-polyacrylamide

gel electrophoresis. Asseen inFig. 5,largeand

medium T-antigens from mutant 45-infected cells migrate ahead of those from wild type.

Small T-antigen, however, is thesamein both

mutant 45-andwild-type-infectedcells.(The37

and62Kprotein bandsarebelievedtobe

anal-ogous tothe37and 55Kspecies that Hutchinson

et al.

[16]

foundbytryptic peptide analysisto

be unrelatedtosmall,medium,orlarge T-anti-gen.) Theprotein band labeled VP1 corresponds in sizetothemajorpolyomaviruscapsid protein, VP1.Cells infected wthasmall-plaquestrain of polyoma virus have an altered VP1 (Fig. 5B).

Size estimates of the mutant 45T-antigens

in-dicate that large T-antigen is 3,000 to 4,000

daltonssmallerthanwildtype andthat medium T-antigen is approximately 2,000 daltons smaller. The size of the deletionin mutant 45 DNA predicts a 2,600-dalton reduction in

pro-tein molecular mass. Chasing the 3S-labeling

medium for 6 or 12 .h with medium containing

an excess ofunlabeled methionine revealed no

significant differences between mutant 45 and

wild-type virus in the amounts andstability of theT-antigens (Fig.5).

Schaffhausen et al. have observed in vivo

phosphorylation of large and medium T-anti-gensandnophosphorylationofsmallT-antigen,

when infected cells are incubated with

[32P]-A B C D E F G H

t1..u K -- tI - .

94K- <*-t-- r Ig-T

68

K-342K-

_*

r * X

=med-T

00 w#m moI-E-VPIW-f

-

42K-____ _-__ _ 4 _-<37K

29K-*Qs ~~~~~sm-T

21.5K _

FIG. 5. Fluorogram of T-antigens labeled with

[36S]methionine.Culturesof3T6 cellswere infected

with Crawford small-plaque polyoma virus

(multi-plicity of infection = 10). The infected cells were

incubated at 33°C for 43 h, labeled for 2 h with

[3Slmethionine (100,iCi/ml), and harvested bythe procedure of Hutchinson et al. (16). In pulse-chase

experiments,cellswerewashed and chased with

me-diumcontaining10times thenormal concentration

of methionine before harvesting.

Immunoprecipita-tionofradiolabeled cellextracts wascarriedoutby

amodifiedStaphylococcus proteinAprocedure (16).

Anti-polyomatumor serum waskindlyprovidedby T. Benjamin. The immuneprecipitateswere electropho-resed through adiscontinuous sodium dodecyl sul-fate-polyacrylamide gel (5 and 12% acrylamide) (20) whichwasthenstained andpreparedfor fluorogra-phy (5). Theproteinmolecularweight markers, phos-phorylase b (94K), bovineserumalbumin(68K), oval-bumin(43K),carbonicanhydrase (29K),andsoybean trypsin inhibitor (21.5K), are indicated on the left. Slot A is from mock-infected cells, slot B is from

Crawfordsmall-plaque-infectedcells, slots C, E, and

Garefromwild-type large-plaque-infected cells, and slotsD, F, andHarefrommutant45-infected cells. SamplesinslotsEand Fwerechasedfor6hbefore harvesting, and those in slots G and H were chased for12h.

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1218

phosphate (27). To determine whether the

al-tered medium andlarge T-antigens frommutant

45 retain their sites for phosphorylation, 3T6

cells were infected and labeled wth

[32P]phos-phate, and the T-antigenswere immunoprecip-itated. Figure 6 shows thatin bothmutant 45

andwild-type viruses,large T-antigen is heavily phosphorylated, whereas medium T-antigen has

alow level ofphosphorylation.

Mutant 45 was tested for defects in known earlygene functions. As mentionedpreviously,

one class ofearly gene mutants, the ts-a

mu-tants, affect a viral function required for the

initiation of viral DNAreplication (9). The rel-ative amounts of viral DNA synthesis in cells infected with either mutant orwild-type virus

wasdetermined byinfecting duplicate plates of

confluent 3T6 cells at amultiplicity of10PFU/

cell. The infected cellswereincubatedat

330C,

labeled with [32P]phosphate from 12 to 36 h postinfection, and harvested via the Hirt

proce-dure.Samplesfrom the Hirtsupematants were

exhaustivelydigested with endo EcoRI and elec-trophoresedthrough1%agarosegels.

Densitom-etertracings ofautoradiogramsof thegelswere

used to calculate the relativeamounts of form

III DNAfrommutant 45-andwild-type-infected

cells. Mutant 45 had slightly lower levels of DNAsynthesis, approximately60% thelevel of

wild type. Although this suggests mutant 45is

slightly impaired in DNAreplication,this defect

hasnosignificanteffectonits growthproperties.

Growth curves at 33 and 39.5°C showed no

significant differences between mutant 45 and wild-type viruses(datanotshown).

Bothclasses ofpreviouslycharacterized early region mutants are defective in virus-induced

cell transformation(3,10).Mutant45 wastested for itsabilitytotransform BHK cellsorFisher

rat3T3 cells toanchorage independenceas

de-terminedby growth in softagar. From the

re-sults of thesetransfornationassays(Table 1),it

appearsthatmutant45transformsasefficiently

aswild-typevirus. Inaddition, preliminary

ex-periments indicate that mutant45 induces

an-giomatous lesions of the liver andlungsofSyrian

hamsters wheninjectedintonewborns.

Although mutant45 isstill abletocarry out

important earlygenefunctionssuch asthe

ini-tiationof viralDNAreplicationand the

trans-formation of

cells,

twoearlygeneproducts,

me-dium andlargeT-antigens,have reduced

molec-ular weights. DNA sequencing of theearly

re-gion,mapping oftheearly mRNA's,andtryptic

peptide analysis of early gene products have permitted the identification of theapproximate

coding regionsfor the three T-antigens (11,30;

R. Kamen, personal communication). As

illus-trated in Fig. 1, the deletion inmutant 45lies

A B C

94K >|

_~-Ig

_-T

68K-:med-T

43K-..._

FIG. 6. Autoradiogram of T-antigens labeled with

[32Plphosphate. Culturesof 3T6 cells were infected withwild-typeandmutant 45viruses (multiplicity of

infection = 10). After 42 h at 33°C, the cells were washed twicewithphosphate-freemedium and then labeledfor4hwith[32P]phosphate(100,Ci/ml).Cell extracts wereprepared and immunoprecipitatedas describedfor35S-labeled cells. The

immunoprecipi-tates were analyzedon a 4 and8%discontinuous sodiumdodecylsulfate-polyacrylamide gel. Thegel

wasstained with Coomassiebrilliantblue,destained,

dried, and autoradiographed. Molecular weight

markers are indicated on the left. Slot A is from

mock-infectedcells,slot B isfromwild type, and slot Cisfrommutant 45.

within thecoding regions for medium andlarge T-antigens and thus would be expectedtoalter mediumand largeT-antigens butnot toaffect smallT-antigen.

The amino acidswithin middle andlarge

T-antigensthataredeleted in mutant45 areshown

in Fig. 4. The most noteworthy sequence of

aminoacids,astretch of sixglutamic acid

resi-dues in middle T-antigen, is just beyond the

limits ofthedeletion.

Severalother viablemutantsofpolyomavirus

with deletions in themiddleof theearlyregion

have recently been reported. Magnusson and

Berg have isolated several mutants with the

deletionsinHpaII-7or -8with lowered

capaci-ties fortransformingrat 1fibroblasts (23).The

precise locationofthese deletionsisnotknown,

however.Griffinand Maddock(13)haveisolated

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TABLE 1. Transformationassays' Transformed

Expt Cell Virus MOI colonies/infected cells

1 BHK Mock 0 0/5.6x104

Wild type 21 73/5.6x104

Mutant 45 61 143/5.6x104

2 BHK Mock 0 0/4.0x104

Wild type 80 94/4.0x 104 Mutant45 70 56/4.0x 104

3 FR3T3 Mock 0 0/2.0x 104

Wild type 60 90/2.0x104

Mutant45 280 296/2.0x 104 aBaby

hamster

kidneycells (BHK-21/13from G. DiMayorca) orFisherrat 3T3celis (FR3T3 from R.

G. Martin) (28) were mock infectedorinfected with

polyoma virus atthe indicatedmultiplicities (MOI).

Infected cells were plated in a layer of 0.33% agar mediumover abaselayerof 0.5% agar medium(22). After28daysat370Cfor BHK cellsor33°Cfor FR3T3 cells, large macroscopically visible colonies were scoredas transformed.

and characterizedtwomutants (dl-8anddl-23)

inthisregion.Thedl-8 mutant mapson oneside

ofmutant 45toward theorigin, andits

approxi-mately100-base-pairdeletionmayslightly

over-lapthe66-base-pair deletion in mutant 45. The

approximately 100-base-pair deletion in dl-23

maps onthe other side of mutant45, but there

is likely to belittle or no overlap between the

deletions in mutants dl-23 and 45. Together,

mutant45,dl-8anddl-23 delete about 250

con-tiguous base pairs (approximately 10% of the

early region). Like mutant45,bothdl-8 and -23

have truncated medium and large T-antigens

and normal-size small T-antigen; however,

un-like mutant 45, dl-8 and dl-23 exhibit altered

replication ortransformation properties. These

viabledeletion mutants constitute a new class of

mutants in theearly regionofthepolyomavirus

genome. Itis of interestthat thisregion shares

nohomologywiththat of simian virus40 orBK

virus (11, 30). It seemsthat their large

T-anti-gens are functional without such a sequence

component.

Wethank PatriciaVoilmerandRoseann White forhelp in preparing thesecondarywholemouseembryocells.Thegift of polyoma anti-T serum from T. Benjamin is gratefully acknowledged.

SupportwasprovidedbyPublicHealthServicegrantCA 13978from the NationalCancerInstitute, training grant GM 07135 toM.M.B.fromthe National Institute ofGeneral Med-icalSciences, andAmericanCancerSociety Faculty Research Award FRA152 toW.R.F.

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1220 NOTES

I.Synthesis ofamodifiedheat-labile T antigen incells

transformed withtheta-amutant.J. Virol.15:393-397. 27. Schaffhausen, B. S.,J.E.Silver,and T. LBenjamin.

1978.Tumorantigen(s)incellsproductively infected by wild-typepolyoma virus andmutantNG-18. Proc.Natl. Acad. Sci. U.S.A. 75:79-83.

28. Seif, R., and F. Cuzin. 1977. Temperature-sensitive growthregulation inonetypeoftransformedratcells induced by thetsa mutantofpolyoma virus. J.Virol.

24:721-728.

29. Silver, J.,B. Schaffhausen, and T. Benjamin.1978. Tumor antigens induced by nontransformingmutants

ofpolyoma virus. Cell15:485-496.

30. Soeda, E., J. R. Arrand, N. Smolar, and B. E. Griffin. 1979.Sequence from earlyregionof polyoma virus DNA containingviral replication origin andencoding small, middle and (part of) large T-antigens. Cell17:357-370.

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